The present invention relates generally to methods for the rapid detection of viable cells involving staining viable cells in a sample with a fluorescent dye and measuring the fluorescence. The invention is more particularly related to methods and kits for the rapid identification of microbes in an environmental sample.
Biowarfare and bioterrorism include the intentional or the alleged use of viruses, bacteria, fungi and toxins to produce death or disease in humans, animals or plants. While biowarfare poses substantial health risks, bioterrorism also possesses the ability to severely disrupt individuals"" lives, as well as societies and economies. Unfortunately, hoaxes and false alarms similarly disrupt lives and work. Individuals affected by both credible and false biowarfare threats are often forced to wait long time periods while officials determine if a credible biological threat exists, even though only one or two out of several hundred responses may result in a credible threat. Clearly, methods of rapidly determining whether a credible biological threat exists are vitally important in preventing both disease and societal and economic disruption.
A variety of methods are currently employed for the detection of microbial biowarfare agents, such as bacteria and yeast. These methods include, but are not limited to, conventional cell viability assays such as the standard plate count, dye reduction and exclusion methods, electrometric techniques, microscopy, flow cytometry, bioluminescence, colorimetry, and turbidity. However, such methods are generally not useful for rapid detection, as required for field testing and rapid determination of the existence of a potential biological threat. These methods typically require highly trained personnel and expensive instrumentation. In addition, many of these assays require substantially pure populations of cells or long incubation periods.
Additional methods specifically designed to detect biological threats include, for example, immunodiagnostic assays, DNA assays using real-time polymerase chain reaction (PCR), mass spectrometry, enhanced bioluminescence assays, protein nanoarrays, volatile organic compound analysis, and multi photon detection. Again, most of these assays require expensive instrumentation and highly-trained personnel, and cannot be rapidly performed in the field.
Several handheld kits for the detection of biowarfare agents have been described. Sensitive Membrane Antigen Rapid Test (SMART(trademark)) Tickets are currently sold commercially for the rapid detection of Bacillus anthracis spores, V. cholerae serotype 01, Yersina pestis, and Botulism toxin. SMART(trademark) Tickets employ a calorimetric immunoassay designed for the direct presumptive recognition or detection of a presumed biowarfare agent(s). However, the analytical sensitivity of these assays is limited by the current technology, and data provided by manufacturers indicate that a minimum of 10,000 spores is required to generate a positive signal. This number of spores would require a relatively heavy contamination of the area (sample) being tested.
Bio Threat Alert(trademark) test strips are currently marketed for the detection of Bacillus anthracis, ricin toxin, Botulinum Toxin, Staphococcal Enterotoxin B, Yersina pestis, and Francisella tularensis. The Bio Threat Alert(trademark) test strip employs agent-specific antibodies to positively identify the potential threat. Screening results are produced in approximately 15 minutes and may be read visually or using the Guardian Bio Threat Alert(trademark) Test Strip Reader.
Although SMART(trademark) Tickets and Bio Threat Alert test strips are designed for rapid field detection of biowarfare agents, their usefulness appears limited by low sensitivity of detection and their ability to detect a very limited number of microbial biowarfare agents. Further, such antibody-dependent tests are susceptible to the possibility of designed or naturally occurring biological mechanisms that would result in an escape from immune detection. For example, several standard dual or multidye fluorescence filter set are known and available from microscope and filter manufacturers including Zeiss, Leica, Chroma Technologies, Nikon and the like.
The present invention for the detection and quantitation of cells is designed to overcome problems that have been identified within the field. The present technology circumvents the need for training personnel in how to plate, grow and count viable cells from colonies on agar plates and eliminates nearly all training and maintenance costs associated with most other microbial biowarfare agent detection and quantification methods. In addition, the invention substantially decreases the time needed for biowarfare agent detection, permitting more accurate detection in less than twenty minutes, while still allowing for the detection and quantification of any live cell present in the test sample. Finally, the technology offers substantial cost savings over most existing methods of microbial biowarfare agent detection and quantitation.
Briefly, the present invention describes methods and kits for detecting and quantifying viable cells in a sample using fluorescent dyes that can be actively internalized by viable cells and have fluorescence properties measurably altered when bound to target components.
In one embodiment of the present invention, a method for quantifying viable cells in a sample is disclosed, comprising the following steps: (1) contacting a sample with a fluorescent dye, wherein the dye is actively or passively internalized by the cells and has fluorescence properties that are measurably altered when bound to target components; (2) detecting total fluorescence of the sample; and (3) comparing the fluorescence produced by the sample to the fluorescence produced by a control substance. Within certain embodiments, (1) the cells in the sample are bacteria, spores, and yeast and other fungi; (2) the fluorescent dye binds to DNA of the viable cells; (3) the sample is treated with DNase before it is mixed with the fluorescent dye; (4) the sample is treated with an agent that affects a cell membrane property of the cells (e.g., a detergent) prior to, subsequently or concurrently with the fluorescent dye; or (5) the fluorescent dye is acridine orange, Hoechst 33258, PicoGreen(trademark), SYTO(copyright) 16, SYBR(copyright) Green I, Texas Red(copyright), Redmond Red(trademark), Bodipy(copyright) Dyes, or Oregon Green(trademark).
In certain embodiments, the cells in the sample are Bacillus anthracis, Bacillus cereus, Clostridium botulinum, Yersinia pestis, Yersinia enterocolitica, Francisella tularensis, Brucella species, Clostridium perfringens, Burkholderia mallei, Burkholderia pseudomallei, Staphylococcus species, Tuberculosis species, Escherichia coli, Group A Streptococcus, Group B streptococcus, Streptococcus pneumoniae, Helicobacter pylori, Francisella tularensis, Salmonella enteritidis, Mycoplasma hominis, Mycoplasma orale, Mycoplasma salivarium, Mycoplasma fermentans, Mycoplasma pneumoniae, Mycobacterium bovis, Mycobacterium tuberculosis, Mycobacterium avium, Mycobacterium leprae, Rickettsia rickettsii, Rickettsia akari, Rickettsia prowazekii, Rickettsia canada, Coxiella burnetti, Aspergillus varieties, Mucor pusillus, Rhizopus nigricans, Candida albicans, C. parapsilosis, C. tropicalis, C. pseudotropicalis, Torulopsis glabrata, Aspergillus niger, Candida dubliniensis, Blastomyces dermatitidis, Coccidioides immitis, Histoplasma capsulatum, Aspergillus species, Candida species, Cryptococcus neoformans, Bacillus subtilis, Bacillus subtilus niger, Bacillus thuringiensis, all Bacillus sp. and/or Sporothrix schenckii. 
The present invention also discloses kits for detecting or quantifying viable cells. One such kit comprises a cell suspension solution, a fluorescent dye that can be actively internalized by viable cells, and instruction for detecting dye binding to cellular components of viable cells. The cell suspension solution may include a DNase or an agent that affects cell membrane property, such as a detergent. In certain embodiments, the fluorescent dye may be acridine orange, Hoechst 33258, PicoGreen(trademark), SYTO(copyright) 16, SYBR(copyright) Green I, Texas Red(copyright), Redmond Red(trademark), Bodipy(copyright) Dyes, or Oregon Green(trademark). Such fluorescent dyes are readily available from commercial sources including Molecular Probes (Eugene, Oreg.), Sigma Chemical (St Louis, Mo.), Amersham (Arlington Heights, Ill.), Callbiochem-Novabiochem (La Jolla, Calif.), Synthetic Genetics (San Diego, Calif.), and the like.
In another embodiment, a kit comprises a first container containing a first solution, means for placement of a sample containing an unknown number of viable cells into a solution containing a fluorescent dye that can be internalized predominately by the viable cells and binds selectively to double-stranded DNA or other specific cellular components, whereupon its fluorescence is altered to a measurable degree, and means for illuminating the mixture of first solution with said sample with excitation light and measuring fluorescence emitted by said mixture, thereby detecting the presence of viable cells in said sample.
In yet another embodiment, a kit comprises a first container containing a first solution, means for placement of a sample containing an unknown number of viable cells into a first solution, means for concentrating the solids and cells from the mixture of said first solution with said sample and retaining said solids from the remainder of said mixture, a second solution containing a fluorescent dye that can be internalized predominately by the viable cells and binds selectively to double-stranded DNA or other specific cellular components, whereupon its fluorescence is altered to a measurable degree, means for mixing said second solution with said solids to form a second mixture, and means for illuminating the mixture of said second solution with said solids with excitation light and measuring fluorescence emitted by said mixture, thereby detecting viable cells in said sample.
Upon binding, the fluorescence properties of the fluorescent dye are altered to a measurable degree. The first solution may contain a DNase or an agent that affects a bacterial cell membrane property, such as a detergent. Within certain embodiments, the fluorescent dye may be acridine orange, Hoechst 33258, PicoGreen(trademark), SYTO(copyright) 16, SYBR(copyright) Green I, Texas Red(copyright), Redmond Red(trademark), Bodipy(copyright) Dyes, or Oregon Green(trademark).